The present invention concerns novel nucleic acid sequences, vectors and host cells containing them, amino acid sequences encoded by said sequences, and antibodies reactive with said amino acid sequences, as well as pharmaceutical compositions comprising any of the above. The present invention further concerns methods for screening for candidate activator or deactivators utilizing said amino acid sequences.
Vascular endothelial growth factor (VEGF) is a heparin-binding growth factor specific for vascular endothelial cells that is able to induce angiogenesis in vivo. DNA sequencing suggests the existence of several molecular species of VEGF. VEGFs are secreted proteins in contrast to other endothelial cell mitogens such as acidic or basic fibroblast growth factors and platelet-derived endothelial cell growth factors. VEGF was found to augment human growth by inducing neovascularization. Thus it was suggested that neutralization of VEGF activity may have clinical application in inhibiting malignant cells-induced angiogenesis, decreasing blood supply to the cancerous tissue, leading eventually to its destruction. VEGF has various other functions on endothelial cells, the most prominent of which is the induction of proliferation and differentiation. It was found to be capable of preventing serum starvation-induced apoptosis and this inhibition may represent a major aspect of the regulatory activity of VEGF on vascular endothelium.
VEGF was also found to be involved in the development and the growth of ovarian corpus luteum (CL), since its development is dependent on the growth of new capillary vessels. It has been reported that Flt-1 receptors which inhibit vascular endothelial growth factor bioactivity, resulted in complete separation of corpus luteum angiogenesis in a rat model of hormonally induced ovulation, indicated that VEGF is essential for CL angiogenesis and may be involved in the control of fertility and treatment of ovarian disorders characterized by hypervascularity and hyperplasia.
The human VEGF gene has been recently assigned to chromosome 6p21.2. cDNA sequence analysis of a variety of human VEGF clones had initially indicated that VEGF may exist as one of four different molecular species, having respectively, 121, 165, 189 and 206 amino acids (VEGF121, VEGF165, BEGF206). Alternative exon splicing of a single VEGF gene is the basis for this molecular heterogeneity, VEGF165 lacks the residues encoded by exon 6, while VEGF121 lacks the residues encoded by exons 6 and 7. VEGF189 has an insertion of 24 amino acids highly enriched in basic residues and VEGF206 has an additional insertion of 17 amino acids. VEGF165 is the predominant isoform secreted by a variety of normal and transformed cells. Transcripts encoding VEGF121 and VEGF189 are detected in the majority of cells and tissues expressing the VEGF gene. In contrast, VEGF206 is a very rare form.
Native VEGF is a basic, heparin-binding, homodimeric glycoprotein of 45 kDA. These properties correspond to those of VEGF165. VEGF121 is an acidic polypeptide that fails to bind to heparin. VEGF189 and VEGF200 are more basic and bind to heparin with greater affinity than VEGF165. VEGF121 is a freely soluble protein; VEGF165 is also secreted, although a significant fraction remains bound to the cell surface and the extracellular matrix (ECM). VEGF189 and VEGF206 are almost completely sequestered in the ECM, but may be released in a soluble form by heparin or heparinase. Also, these long forms may be released by plasmin following cleavage at the COOH terminus (Ferrara, N., European J. of Cancer, 32A(14):2413-2422 (1996)).
In the following description and claims use will be made, at times, with a variety of terms, and the meaning of such terms as they should be construed in accordance with the invention is as follows:
xe2x80x9cVascular endothelial growth factor variant (VEGFV) nucleic acid sequencexe2x80x9dxe2x80x94the sequence shown in SEQ ID NO: 1, sequences having at least 70% identity to said sequence and fragments of the above sequences of least 20 b.p. long. This sequence is a sequence coding for a novel alternative splice variant of the native VEGF. While the known VEGF peptides include 206, 189, 105 or 121 amino acids, the novel VEGF variant peptide of the invention includes only 141 amino acids, xe2x88x9227 of which being in the signal peptide and 114 being present in the mature protein. According to the terminology used in the publication of Ferrara (supra) this new variant should be termed VEGF114.
xe2x80x9cVascular endothelial growth factor variant (VEGFV product)xe2x80x94also referred at times as the xe2x80x9cVEGFV proteinxe2x80x9d or xe2x80x9cVEGFV polypeptidexe2x80x9dxe2x80x94is an amino acid sequence having the first 141 amino acids of the native VEGF. This naturally occurring sequence is the result of alternative splicing. The amino acid sequence may be a peptide, a protein, as well as peptides or proteins having chemically modified amino acids (see below) such as a glycopeptide or glycoprotein. An example of an VEGFV product is shown in SEQ ID NO: 2. The term also includes analogues of said sequences in which one or more amino acids has been added, deleted, substituted (see below) or chemically modified (see below) as well as fragments of this sequence having at least 10 amino acids.
xe2x80x9cNucleic acid sequencexe2x80x9dxe2x80x94a sequence composed of DNA nucleotides, RNA nucleotides or a combination of both types and may includes natural nucleotides, chemically modified nucleotides and synthetic nucleotides.
xe2x80x9cAmino acid sequencexe2x80x9dxe2x80x94a sequence composed of any one of the 20 naturally appearing amino acids, amino acids which have been chemically modified (see below), or composed of synthetic amino acids.
xe2x80x9cFragment of VEGFV productxe2x80x9dxe2x80x94a polypeptide which has an amino acid sequence which is the same as part of but not all of the amino acid sequence of the VEGFV product.
xe2x80x9cFragments of VEGFV nucleic acid sequencexe2x80x9d a continuous portion, preferably of about 20 nucleic acid sequences of the VEGFV nucleic acid sequence.
xe2x80x9cConservative substitutionxe2x80x9dxe2x80x94refers to the substitution of an amino acid in one class by an amino acid of the same class, where a class is defined by common physicochemical amino acid side chain properties and high substitution frequencies in homologous proteins found in nature, as determined, for example, by a standard Dayhoff frequency exchange matrix or BLOSUM matrix. [Six general classes of amino acid side chains have been categorized and include: Class I (Cys); Class II (Ser, Thr, Pro, Ala, Gly); Class III (Asn, Asp, Gln, Glu); Class IV (His, Arg, Lys); Class V (Ile, Leu, Val, Met); and Class VI (Phe, Tyr, Trp). For example, substitution of an Asp for another class III residue such as Asn, Gln, or Glu, is a conservative substitution.
xe2x80x9cNon-conservative substitutionxe2x80x9dxe2x80x94refers to the substitution of an amino acid in one class with an amino acid from another class; for example, substitution of an Ala, a class II residue, with a class III residue such as Asp, Asn, Glu, or Gln.
xe2x80x9cChemically modifiedxe2x80x9dxe2x80x94when referring to the product of the invention, means a product (protein) where at least one of its amino acid residues is modified either by natural processes, such as processing or other post-translational modifications, or by chemical modification techniques which are well known in the art. Among the numerous known modifications typical, but not exclusive examples include: acetylation, acylation, amidation, ADP-ribosylation, glycosylation, GPI anchor formation, covalent attachment of a lipid or lipid derivative, methylation, myristlyation, pegylation, prenylation, phosphorylation, ubiqutination, or any similar process.
xe2x80x9cBiologically activexe2x80x9dxe2x80x94refers to the VEGFV product having structural, regulatory or biochemical functions of the naturally occurring VEGFV product, for example the same effect on vascular endothelial cells.
xe2x80x9cImmunologically activexe2x80x9d defines the capability of a natural, recombinant or synthetic VEGFV product, or any fragment thereof, to induce a specific immune response in appropriate animals or cells and to bind with specific antibodies. Thus, for example, a biologically active fragment of VEGFV product denotes a fragment which retains some or all of the immunological properties of the VEGFV product, e.g can bind specific anti-VEGFV product antibodies or which can elicit an immune response which will generate such antibodies or cause proliferation of specific immune cells which produce VEGFV.
xe2x80x9cOptimal alignmentxe2x80x9dxe2x80x94is defined as an alignment giving the highest percent identity score. Such alignment can be performed using a variety of commercially available sequence analysis programs, such as the local alignment program LALIGN using a ktup of 1, default parameters and the default PAM. A preferred alignment is the one performed using the CLUSTAL-W program from MacVector (TM), operated with an open gap penalty of 10.0, an extended gap penalty of 0.1, and a BLOSUM similarity matrix. If a gap needs to be inserted into a first sequence to optimally align it with a second sequence, the percent identity is calculated using only the residues that are paired with a corresponding amino acid residue (i.e., the calculation does not consider residues in the second sequences that are in the xe2x80x9cgapxe2x80x9d of the first sequence).
xe2x80x9cHaving at least X% identityxe2x80x9dxe2x80x94with respect to two amino acid or nucleic acid sequence sequences, refers to the percentage of residues that are identical in the two sequences when the sequences are optimally aligned. Thus, 70% amino acid sequence identity means that 70% of the amino acids in two or more optimally aligned polypeptide sequences are identical.
xe2x80x9cIsolated nucleic acid molecule having an VEGFV nucleic acid sequencexe2x80x9dxe2x80x94is a nucleic acid molecule that includes the coding VEGFV nucleic acid sequence. Said isolated nucleic acid molecule may include the VEGFV nucleic acid sequence as an independent insert; may include the VEGFV nucleic acid sequence fused to an additional coding sequences, encoding together a fusion protein in which the VEGFV coding sequence is the dominant coding sequence (for example, the additional coding sequence may code for a signal peptide); the VEGFV nucleic acid sequence may be in combination with non-coding sequences, e.g., introns or control elements, such as promoter and terminator elements or 5xe2x80x2 and/or 3xe2x80x2 untranslated regions, effective for expression of the coding sequence in a suitable host; or may be a vector in which the VEGFV protein coding sequence is a heterologous.
xe2x80x9cExpression vectorxe2x80x9dxe2x80x94refers to vectors that have the ability to incorporate and express heterologous DNA fragments in a foreign cell. Many prokaryotic and eukaryotic expression vectors are known and/or commercially available. Selection of appropriate expression vectors is within the knowledge of those having skill in the art.
xe2x80x9cDeletionxe2x80x9dxe2x80x94is a change in either nucleotide or amino acid sequence in which one or more nucleotides or amino acid residues, respectively, are absent.
xe2x80x9cInsertionxe2x80x9d or xe2x80x9cadditionxe2x80x9dxe2x80x94is that change in a nucleotide or amino acid sequence which has resulted in the addition of one or more nucleotides or amino acid residues, respectively, as compared to the naturally occurring sequence.
xe2x80x9cSubstitutionxe2x80x9dxe2x80x94replacement of one or more nucleotides or amino acids by different nucleotides or amino acids, respectively. As regards amino acid sequences the substitution may be conservative or non-conservative.
xe2x80x9cAntibodyxe2x80x9dxe2x80x94refers to IgG, IgM, IgD, IgA, and IgG antibody. The definition includes polyclonal antibodies or monoclonal antibodies. This term refers to whole antibodies or fragments of the antibodies comprising the antigen-binding domain of the anti-VEGFV product antibodies, e.g. antibodies without the Fc portion, single chain antibodies, fragments consisting of essentially only the variable, antigen-binding domain of the antibody, etc.
xe2x80x9cActivatorxe2x80x9dxe2x80x94as used herein, refers to a molecule which mimics the effect of the natural VEGFV product or at times even increases or prolongs the duration of the biological activity of said product, as compared to that induced by the natural product. The mechanism may be by binding to the VEGFV receptor, by prolonging the lifetime of the VEGFV, by increasing the activity of the VEGFV on its target (vascular endothelial cells), by increasing the affinity of VEGFV to its receptor, etc. Activators may be polypeptides, nucleic acids, carbohydrates, lipids, or derivatives thereof, or any other molecules which can bind to and activate the VEGFV product.
xe2x80x9cDeactivatorxe2x80x9d or (xe2x80x9cInhibitorxe2x80x9d)xe2x80x94refers to a molecule which modulates the activity of the VEGFV product in an opposite manner to that of the activator, by decreasing or shortening the duration of the biological activity of the VEGFV product. This may be done by blocking the binding of the VEGFV to its receptor (competitive or non-competitive inhibition), by causing rapid degradation of the VEGFV, etc. Deactivators may be polypeptides, nucleic acids, carbohydrates, lipids, or derivatives thereof, or any other molecules which bind to and modulate the activity of said product.
xe2x80x9cTreating a diseasexe2x80x9dxe2x80x94refers to administering a therapeutic substance effective to ameliorate symptoms associated with a disease, to lessen the severity or cure the disease, or to prevent the disease from occurring.
xe2x80x9cDetectionxe2x80x9dxe2x80x94refers to a method of detection of a disease. This term may refer to detection of a predisposition to a disease.
xe2x80x9cProbexe2x80x9dxe2x80x94the VEGFV nucleic acid sequence, or a sequence complementary therewith, when used to detect presence of other similar sequences in a sample. The detection is carried out by identification of hybridization complexes between the probe and the assayed sequence. The probe may be attached to a solid support or to a detectable label.
The present invention is based on the surprising finding that there exist in humans a novel variant of the VEGF protein, having 141 amino acid (114 amino acids of the mature protein without the signal peptide) than the known VEGF. The nucleic sequence coding for this variant was identified as being from the same locus as the known VEGF and thus it was concluded that the variant is not encoded from a different gene than the known VEGF, but is the result of alternative splicing of the known VEGF.
Thus the present invention provides by its first aspect, a novel isolated nucleic acid molecule comprising or consisting of the coding sequence SEQ ID NO: 1, fragments of said coding sequence having at least 20 nucleic acids, or a molecule comprising a sequence having at least 70%, preferably 80%, and most preferably 90% identity to SEQ ID NO:1.
The present invention further provides a protein or polypeptide comprising or consisting of an amino acid sequence encoded by any of the above nucleic acid sequences, termed herein xe2x80x9cVEGFV productxe2x80x9d, for example, an amino acid sequence having the sequence as depicted in SEQ ID NO: 2, fragments of the above amino acid sequence having a length of at least 10 amino acids, as well as homologs of the amino acid sequences SEQ ID NO.:2 in which one or more of the amino acid residues has been substituted (by conservative or non-conservative substitution) added, deleted, or chemically modified.
The present invention further provides nucleic acid molecule comprising or consisting of a sequence which encodes the above amino acid sequences, (including the fragments and analogs of the amino acid sequences). Due to the degenerative nature of the genetic code, a plurality of alternative nucleic acid sequences, beyond SEQ ID NO:1, can code for the amino acid sequence of the invention. Those alternative nucleic acid sequences which code for the same amino acid sequences codes by the sequence SEQ ID NO: 1 are also an aspect of the of the present invention.
The present invention further provides expression vectors and cloning vectors comprising any of the above nucleic acid sequences, as well as host cells transfected by said vectors.
The present invention still further provides pharmaceutical compositions comprising, as an active ingredient, said nucleic acid molecules, said expression vectors, or said protein or polypeptide.
These pharmaceutical compositions are suitable for the treatment of diseases and pathological conditions, which can be ameliorated or cured by raising the level of the VEGFV product.
By a second aspect, the present invention provides a nucleic acid molecule comprising or consisting of a non-coding sequence which is complementary to that of SEQ ID NO:1, or complementary to a sequence having at least 70% identity to said sequence or a fragment of said two sequences. The complementary sequence may be a DNA sequence which hybridizes with the SEQ of ID NO:1 or hybridizes to a portion of that sequence having a length sufficient to inhibit the transcription of the complementary sequence. The complementary sequence may be a DNA sequence which can be transcribed into an mRNA being an antisense to the mRNA transcribed from SEQ ID NO:1 or into an mRNA which is an antisense to a fragment of the mRNA transcribed from SEQ ID NO:1 which has a length sufficient to hybridize with the mRNA transcribed from SEQ ID NO: 1, so as to inhibit its translation. The complementary sequence may also be the mRNA or the fragment of the mRNA itself.
The nucleic acids of the second aspect of the invention may be used for therapeutic or diagnostic applications for example for detection of the expression of VEGFV. The proportion of expression of the VEGF variant of the present invention as compared to the known VEGF variants may be indicative to a variety of physiological or pathological conditions.
The present invention also provides expression vectors comprising any one of the above defined complementary nucleic acid sequences and host cells transfected with said nucleic acid sequences or vectors, being complementary to those specified in the first aspect of the invention.
The invention also provides anti-VEGFV product antibodies, namely antibodies directed against the VEGFV product which specifically bind to said VEGFV product. Said antibodies are useful both for diagnostic and therapeutic purposes. For example said antibody may be as an active ingredient in a pharmaceutical composition as will be explained below.
The present invention also provides pharmaceutical compositions comprising, as an active ingredient, the nucleic acid molecules which comprise or consist of said complementary sequences, or of a vector comprising said complementary sequences. The pharmaceutical composition thus provides pharmaceutical compositions comprising, as an active ingredient, said anti-VEGFV product antibodies.
The pharmaceutical compositions comprising said anti-VEGFV product antibodies or the nucleic acid molecule comprising said complementary sequence, are suitable for the treatment of diseases and pathological conditions where a therapeutically beneficial effect may be achieved by neutralizing the VEGFV or decreasing the amount of the VEGFV product or blocking its binding to the receptor, for example, by the neutralizing effect of the antibodies, or by the decrease of the effect of the antisense mRNA in decreasing expression level of the VEGFV product.
According to the third aspect of the invention the present invention provides methods for detecting the level of the transcript (mRNA) of said VEGFV product in a body fluid sample, or in a specific tissue sample, for example by use of probes comprising or consisting of said coding sequences; as well as methods for detecting levels of expression of said product in tissue, e.g. by the use of antibodies capable of specifically reacting with the above amino acid sequences. Detection of the level of the expression of the VEGF variant of the invention in particular as compared to that of the known VEGF variants may be indicative of a plurality of physiological or pathological conditions.
The method, according to this latter aspect, for detection of a nucleic acid sequence which encodes the VEGFV product in a biological sample, comprises the steps of:
(a) providing a probe comprising at least one of the nucleic acid sequence defined above;
(b) contacting the biological sample with said probe under conditions allowing hybridization of nucleic acid sequences thereby enabling formation of hybridization complexes;
(c) detecting hybridization complexes, wherein the presence of the complex indicates the presence of nucleic acid sequence encoding the VEGFV product in the biological sample.
By a preferred embodiment the probe is part of a nucleic acid chip used for detection purposes, i.e. the probe is a part of an array of probes each present in a known location on a solid support.
The nucleic acid sequence used in the above method may be a DNA sequence an RNA sequence, etc; it may be a coding or a sequence or a sequence complementary thereto (for respective detection of RNA transcripts or coding-DNA sequences). By quantization of the level of hybridization complexes and calibrating the quantified results it is possible also to detect the level of the transcript in the sample.
Methods for detecting mutations in the region coding for the VEGFV product are also provided, which may be methods carried-out in a binary fashion, namely merely detecting whether there is any mismatches between the normal VEGFV nucleic acid sequence and the one present in the sample, or carried-out by specifically detecting the nature and location of the mutation.
The present invention also concerns a method for detecting VEGFV product in a biological sample, comprising the steps of:
(a) contacting with said biological sample the antibody of the invention, thereby forming an antibody-antigen complex; and
(b) detecting said antibody-antigen complex
wherein the presence of said antibody-antigen complex correlates with the presence of VEGFV product in said biological sample.
By yet another aspect the invention also provides a method for identifying candidate compounds capable of binding to the VEGFV product and modulating its activity (being either activators or deactivators). The method includes:
(i) providing a protein or polypeptide comprising an amino acid sequence substantially as depicted in SEQ ID NO: 2, or a fragment of such a sequence;
(ii) contacting a candidate compound with said amino acid sequence;
(iii) measuring the physiological effect of said candidate compound on the activity of the amino acid sequences and selecting those compounds which show a significant effect on said physiological activity.
The activity of the amino acid which should be changed by the modulator (being either the activator or deactivator) may be for example the binding of the VEGFV product to its native receptor, effect on the modulation in the effect of VEGFV on vascular endothelial cells, etc. Any modulator which changes such an activity has an intersecting potential.
The present invention also concerns compounds identified by the above methods described above, which compound may either be an activator of the serotonin-receptor like product or a deactivator thereof.